ARF is the second most commonly inactivated gene in human cancer. It prevents oncogenic transformation by activating p53-dependent growth arrest or apoptosis, primarily by sequestering the Mdm2 oncogene in nucleoli and blocking Mdm2-mediated degradation of p53. Recent findings indicate that 1) other factors are required for p53-dependent growth arrest, and 2) ARF can inhibit growth through other pathways that do not involve p53 or Mdm2. This proposal aims to identify and characterize additional mediators and inhibitors of ARF-induced growth arrest. The ultimate objective is to delineate mechanisms of growth suppression by ARF. Aim 1 will determine the role of identified ARF binding proteins, Mdm2 and two novel proteins (Parfs) that associate with ARF in nucleoli, in ARF-mediated growth suppression. The mechanism by which Mdm2 antagonizes p53-independent activities of ARF will be defined by assaying ARF function in p53/Mdm2-null cells expressing Mdm2 mutants. Interaction domains within Parfs and ARF will be identified through mutagenesis, while the hypothesis that Parfs are growth inhibitors required for ARF-induced growth arrest will be tested in cells which overexpress or lack functional Parfs. Aim 2 will define the role of two ARE-associated phosphoproteins, p58 and p65, in ARF signaling pathways. We will test if p58 and p65 are phosphorylated in response to ARF and if they selectively bind to growth inhibitory forms of ARF in a p53-dependent manner. The identities of p58 and p65 will be determined by testing if p58 is the 58 kDa form of Mdm2 (since it is a phosphoprotein) and biochemically purifying ARF-associated proteins of 50-70 kDa by affinity chromatography. In Aim 3, an unbiased genetic screen using retroviral cDNA libraries will be performed to identify and characterize novel genes that override p53-dependent and p53-independent ARF-induced arrest. These studies will advance our fundamental understanding of ARF function in the different signaling pathways, and as such, provide knowledge that is relevant to developing appropriate anticancer strategies.